Abstract Metastasis is responsible for the majority of cancer deaths despite progress in cancer treatment over the past decades. The PI3K signaling pathway, which is mutated at high frequencies in various forms of human cancers, has been implicated in the metastatic process, but the cellular mechanism is poorly understood. Recent studies into the fundamental mechanism of cell migration open up new opportunities to investigate how cancer cells migrate and metastasize. Using Dictyostelium and human neutrophils as models, the principle investigator (PI) has demonstrated that cell migration depends on the coupling between an excitable Ras-PI3K network and a cytoskeletal network to drive the formation of protrusions. The objectives of this proposal are (1) to assess to what extent a similar coupling between an excitable PI3K signaling network and a cytoskeletal network drives the migration of cancer cells; (2) to investigate how altered PI3K signaling activity affect the dynamics of the molecular networks to change the motility and metastatic behavior of cancer cells. To achieve these objectives, the PI will use cutting edge imaging techniques to visualize the spatiotemporal dynamics of various signaling and cytoskeletal activities in breast cancer cells to understand the features and functions of these activities (Aim 1). The PI3K signaling activity will be altered using genetic or pharmacological methods to study how the signaling and cytoskeletal activities are affected, and how these changes affect the migration of cells in vitro (Aim 2). The dynamics of the molecular networks in various cancer cells with or without altered Pi3K signaling will then be correlated with their ability to metastasize in mice (Aim 3). Finally, the PI3K signaling and cytoskeletal activities will be examined in primary and metastatic tumors from breast cancer patients to confirm the clinical relevance of experimental findings (Aim 4). The successful completion of these aims will provide valuable information for developing molecular interventions for metastatic cancers. The PI has received clinical training prior to his research career. The award will support the PI to obtain preliminary results required for independent funding and acquire new skills through collaboration, particularly regarding the use of mice and patient samples. The long-term goal of the PI is to apply fundamental cell biology to the development of new interventions for cancers.